1. Field of the Invention
The present invention relates to the use of a novel simulation technology to rationally optimize the locations and sizes of attached polymeric moieties for modification of proteins, especially therapeutic proteins.
2. Background of the Prior Art
Polymers, and particularly polyethylene glycol (PEG), are highly flexible and soluble and have gained widespread scientific and regulatory acceptance as a chemical modification for therapeutic proteins. For example, PEG attachment (PEGylation) improves PK predominantly by increasing the effective size of a protein, with most significant effects for proteins smaller than 70 kD. PEGylation can also reduce immunogenicity and aggregation. While a variety of chemistries exist for coupling PEGs of various sizes to proteins, the greatest attachment specificity generally arises from PEGylation at the N-terminus or unpaired cysteines. For further information about PEGylation, see for example Roberts, M. J. et al. (2002) “Chemistry for peptide and protein PEGylation” Adv. Drug Deliv. Rev. 54, 459-476 and Kinstler, O. et al. (2002) “Mono-N-terminal poly(ethylene glycol)-protein conjugates” Adv. Drug Deliv. Rev. 54.
Several PEGylated protein therapeutics are currently on the market or in late-stage clinical trials. Schering-Plough's PEG-Intron® (peginterferon alfa-2b) and Roche's PEGasys® (peginterferon alfa-2a), both PEGylated variants of interferon-a (IFNa) used to treat hepatitis C, show significantly improved in vivo efficacy relative to the parent molecules.
One disadvantage of many PEGylated protein therapeutics is that they have significantly reduced specific activity relative to the unmodified proteins (see for example Bailon, P. et al. (2001) “Rational design of a potent, long-lasting form of interferon: a 40-kDa-branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C” Bioconjug. Chem. 12, 195-202; and Wang, Y. S. et al. (2002) “Structural and biological characterization of PEGylated recombinant interferon alpha-2b and its therapeutic implications” Adv. Drug Deliv. Rev. 54, 547-570). Since IFNa is a relatively small protein that contains two receptor-binding interfaces, it is not surprising that a random attachment strategy leads to a decrease in activity. Thus, although PEG attachment is generally useful for improving pharmacokinetics, it often does so at the expense of specific activity. As a result, developers of PEGylated therapeutics are often faced with the difficult challenge of seeking PEG attachment sites that minimally impact the specific activity of the modified protein.
Discovery of optimal PEGylation sites is usually accomplished empirically, requiring extensive experimentation to compare the effects of various PEGylation sites and sizes on the activity of a protein. While some attempts have been made to understand the relationship between attachment site, PEG size, and specific activity of the modified protein, such attempts have rarely yielded accurate predictions. Hence, there is a need in the field for a method that can more accurately predict the relationship among PEG attachment sites, sizes, and specific activities of the resulting proteins.